Centrifugation affects the purity of liquid biopsy-based tumor biomarkers

Centrifugation Affects the Purity of Liquid

Biopsy-Based Tumor Biomarkers

Linda G. Rikkert,

* Edwin van der Pol,

Ton G. van Leeuwen,

Rienk Nieuwland,

Frank A.W. Coumans

Abstract

Biomarkers in the blood of cancer patients include circulating tumor cells (CTCs), tumor-educated platelets (TEPs), tumor-derived extracellular vesicles (tdEVs), EV-associated miRNA (EV-miRNA), and circulating cell-free DNA (ccfDNA). Because the size and density of biomarkers differ, blood is centrifuged to isolate or concentrate the biomarker of interest. Here, we applied a model to estimate the effect of centrifugation on the purity of a biomarker according to published protocols. The model is based on the Stokes equation and was validated using polystyrene beads in buffer and plasma. Next, the model was applied to predict the biomarker behavior during centrifugation. The result was expressed as the recovery of CTCs, TEPs, tdEVs in three size ranges (1–8, 0.2–1, and 0.05–0.2 μm), EV-miRNA, and ccfDNA. Bead recovery was predicted with errors <18%. Most notable cofounders are the 22% contamination of 1–8 μm tdEVs for TEPs and the 8–82% contamination of <1 μm tdEVs for ccfDNA. A Stokes model can predict biomarker behavior in blood. None of the evaluated protocols pro-duces a pure biomarker. Thus, care should be taken in the interpretation of obtained results, as, for example, results from TEPs may originate from co-isolated large tdEVs and ccfDNA may originate from DNA enclosed in <1μm tdEVs. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

Key terms

biomarker; centrifugation; circulating cell-free DNA; circulating tumor cells; exosomes; extracellular vesicles; flow cytometry; microparticles; miRNA; tumor-educated platelets

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(CTCs) (1–3), tumor-educated platelets (TEPs) (4), tumor-derived extracellular vesicles (tdEVs) (5), EV-miRNAs (6), and circulating cell-free DNA (ccfDNA) (7,8). Typically, the first step to isolate a biomarker involves (differential) centrifugation, which isolates particles based on size and density. After centrifugation and optionally further processing, the biomarker is measured based on antigen exposure (CTCs and tdEVs) or on the com-position of RNA (TEPs and EV-miRNAs) or DNA (ccfDNA). Fractions containing the biomarker of interest may be impure and contain substantial quantities of “contami-nants”, for example, other biomarkers. These contaminants may affect the obtained signal and potentially lead to the misinterpretation of results. Therefore, we applied a model based on the Stokes equation to assess the purity of biomarkers after centrifugation according to protocols used by other groups to study their biomarkers of interest (1–8).

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Stokes Model

Figure 1 shows the principle of centrifugation. Before centrifugation, particles are uniformly distributed in a medium. Upon centrifugation, particles denser than the medium will travel toward the bottom of the tube. After centrifugation, the top 1

Medical Cell BioPhysics, University of Twente, Enschede, the Netherlands

2

Amsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, the Netherlands

3

Amsterdam UMC, University of Amsterdam, Vesicle Observation Center, Amsterdam, the Netherlands

4

Amsterdam UMC, University of Amsterdam, Biomedical Engineering and Physics, Amsterdam, the Netherlands

Received 27 July 2018; Revised 28 September 2018; Accepted 1 October 2018

Grant sponsor: Netherlands Organisation for Scientific Research—Domain Applied and Engineering Sciences (NWO-TTW), Grant numberVENI 15924, Grant numberPerspectief CANCER-ID 14198, Grant numberVENI 13681 Additional Supporting Information may be found in the online version of this article.

*_{Correspondence to: Linda G. Rikkert;}

Amster-dam UMC, University of AmsterAmster-dam, Laboratory of Experimental Clinical Chemistry, Meiberg-dreef 9, Amsterdam 1105 AZ, the Netherlands. Email: l.g.rikkert@amc.uva.nl

Published online in Wiley Online Library (wileyonlinelibrary.com)

DOI: 10.1002/cyto.a.23641

© 2018 The Authors. Cytometry Part A pub-lished by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

fraction is collected, defined here as the “supernatant.” We define the“pellet” as the fraction remaining in the tube. The dashed line in Figure 1 is the interface between the superna-tant and the pellet. The recovery of particles in the pellet is the fraction of particles in the pellet after centrifugation. This recovery depends on the starting point of the particle in the medium, as well as the densities and diameters of the parti-cles. Consequently, the pellet will contain a high concentra-tion of large and high density particles compared to the supernatant. For a swing-out rotor, the distance traveled by a particle can be modeled by the Stokes equation. In the Stokes equation, the speed of the particle results from the balance between the buoyant force and the drag force on a particle in a medium. In a centrifuge, the gravitational acceleration g depends on the distance from the axis of rotation.

g¼ Rω2 _ð1Þ

where R is the distance to the axis of rotation andω is the angular velocity of the centrifuge. After time T, the dis-tance R of a particle starting at R0 is given by the following

equation:

RT¼ R0e

d2ðρP −ρMÞ

18 Sη ω2T ð2Þ

where d and ρP are the diameter and the volumetric

mass density of the particle andρMandη are the volumetric

mass density and viscosity of the medium. To allow nonsphe-rical shapes such as platelets, the shape factor S is introduced (S = 1 for spheres). S accounts for the additional drag these nonspherical particles experience (9). Note that for non-spheres, d is the diameter of a sphere with equivalent volume. Model assumptions are (1) negligible wall effects, (2) instanta-neous formation of the packed cell fraction during centrifuga-tion of whole blood, and (3) aspiracentrifuga-tion sufficiently gentle not to disturb the pellet (see (10,11) for a complete description of the assumptions in the Stokes model). The model was pro-grammed in Matlab (v2017a; MathWorks, Natick, MA) and applied to the evaluated centrifugation protocols.

Stokes Model Sample Properties

Table 1 shows the assumed sample properties, which were derived from the literature for a temperature of 20C (see Supporting Information Table S1 for additional back-ground). If available, volumetric mass densities were taken from references that applied density media with neutral osmotic effects. Platelets have a log-normal volume distribu-tion (12) and a shape factor S of 2.0 (9,13). Platelets can have a volumetric mass density of 1.05–1.09 g/ml, where the lower density is associated with platelets that have secreted their α-granule content but with an unaffected platelet volume dis-tribution (14,15). Most EVs have a diameter of <200 nm, which means that the volumetric mass density is substantially affected by the membrane density. A model describing EVs as cytoplasm enclosed by a cell-like membrane (16) leads to a size-dependent volumetric mass density. For EVs 100, 200, and 1,000 nm, the density is 1.099, 1.081, and 1.064 g/ml, respectively, which is in fair agreement with literature esti-mates between 1.08 and 1.11 g/ml (17–21). Finally, the mass density of ccfDNA is only available in cesium chloride media and the length of ccfDNA is unknown. Assuming a length of <1,000 base pairs, the ccfDNA would be smaller than 50 nm. Model Validation

To validate the model, polystyrene beads were diluted in phosphate-buffered saline (PBS) or blood plasma. The con-centration of beads was measured by flow cytometry before and after centrifugation at 300g for 20 min or 2,700g for 22 min using a Rotina 380R centrifuge (Hettich, Tuttlingen, Germany) or 15,800g for 60 min using a SW 41 Ti rotor and Optima L-80 XP ultracentrifuge (Beckman Coulter, Fullerton, CA), all at 20C and with deceleration set to the minimum possible value. Polystyrene beads were 400, 799, 994, and 3,005 nm in diameter (Thermo Fisher Scientific, Waltham, MA) with a density of 1.05 g/ml. PBS (154 mM NaCl, 1.24 mM Na2HPO42H2O, 0.2 mM NaH2PO42H2O; pH 7.4)

was 0.22 μm filtered (Merck, Darmstadt, Germany). Blood from anonymous healthy donors was obtained after written informed consent in accordance with the Dutch regulations and approved by the medical–ethical assessment committee of the Academic Medical Center, University of Amsterdam. Whole blood was drawn using a 21G needle in EDTA

Figure 1. Centrifugation to isolate biomarker of interest from whole blood. The position of a particle after centrifugation can be described with the stokes equation (Eq. 2). Centrifugation of particles that are uniformly distributed throughout the sample causes large, high-density particles to move down to the pellet (below the dashed line), while small, low-density particles stay in the supernatant (above the dashed line). [Color figure can be viewed at wileyonlinelibrary.com]

vacutainers (BD Biosciences, San Jose, CA) and processed within 15 min after collection. Plasma was obtained from whole blood by double centrifugation at 2,500g for 15 min at 20C using a Rotina 380R centrifuge. The supernatant was pooled between centrifugation steps and pooled before mixing with the polystyrene beads. The concentration of beads before and after centrifugation was measured on side scatter of a flow cytometer (FACSCalibur; BD, Franklin Lakes, NJ, 15 mW 488 nm laser, flow rate ~60 μl/min calibrated by weight (22), SSC 400 V, gain 0, threshold 0), and data were analyzed using FlowJo (v10; FlowJo LLC, Ashland, OR).

Predicted Performance of Centrifugation Protocols We modeled the protocols as described in the literature and shown in Table 2. The dimensions of the low-speed cen-trifugation tube were derived from the 57.462 Sarstedt centri-fugation tube and for ultracentricentri-fugation from the 344059 Beckman Coulter centrifugation tube. For the model, we assume that centrifugation <10,000g is performed using a Rotina 380R centrifuge and centrifugation >10,000g using a SW41 Ti rotor and Optima L-80 XP ultracentrifuge. For most centrifuges, the set g-force is the force at the bottom of the tube. The force elsewhere is given by Eq. 1. This means that a

Table 1. Stokes model sample properties

TYPE AND PROPERTY VALUE USED IN MODEL REFERENCES

CTCs

Density (g/ml) 1.053 (24,25)

Diameter (μm) Uniform. 8–20 (26)

Platelets

Density (g/ml) Norm., mean 1.069, SD 0.0053 (14,15,27)

Diameter (μm) Lognorm., median 2.4, SD 0.6 (12,28)

Shape factor 2.0 (9,13)

EVs

Density (g/ml) Core 1.060, 8 nm shell 1.155 (17–21)

Diameter (μm) Uniform. 0.05–1 ccfDNA Density (g/ml) 1.7 (16,29) Diameter (μm) ≤0.05 (16) Polystyrene beads Density (g/ml) 1.05 MFG Diameter (μm) MFG PBS Density (g/ml) 1.004 (30) Viscosity (mPa s) 1.193 (30) Plasma Density (g/ml) 1.0253 (31,32) Viscosity (mPa s) 1.75 (33–35)

Values at 20C (see Supporting Information Table S1 for more details). Lognorm., lognormal distribution; MFG, manufacturer speci-fications; Norm., normal distribution; SD, standard deviation; Uniform., uniform distribution.

Table 2. Modeled centrifugation protocols

PROTOCOL

BLOOD VOLUME AND

VACUTAINER PROTOCOL SUMMARY REFERENCES

CTCs/tdEVs 10 ml CellSearch WB 7.5 ml:6.5 ml PBS 800g, 10 min! (10) Pel (1–3,5) TEPs 6 ml EDTA WB 120g, 20 min! (3) Sup 360g, 20 min ! (10) Pel, wash 2× (4) EV-miRNA 6 ml EDTA WB 900g, 7 min! (5) Sup 2,500g, 10 min ! (5) Sup 500g,

10 min! (5) Sup

(6) ccfDNA

(Speicher)

10 ml PAXgene ccfDNA WB 200g, 10 min, 1,600g, 10 min! (3) Sup 1,600g, 10 min ! (3) Sup (8) ccfDNA

(Dawson)

10 ml EDTA WB 820g, 10 min! (5) Sup 20,000g, 10 min ! (10) Sup (7)

ccfDNA, circulating cell-free DNA; CTCs, circulating tumor cells; EV-miRNAs, extracellular vesicle-associated miRNAs; TEPs, tumor-educated platelets; tdEVs, tumor-derived extracellular vesicles; WB, whole blood;! (xx) Pel, collect pellet xx mm above bottom of tube or buffy coat (for WB);! (xx) Sup, collect supernatant xx mm above bottom of tube or buffy coat (for WB).

10 cm high sample in a Rotina centrifuge set to 2,500g, has a g-force of 900g at the top of the sample. The same centrifuga-tion protocol on a rotor with a larger diameter will increase the g-force at the top of the sample and thus result in an increased recovery in the pellet. However, we did not model multiple rotor diameters, because most rotors suitable for large tubes have diameters comparable to the Rotina 380R. Results are expressed as the % recovery of CTCs, TEPs, tdEVs in three size ranges from large (1–8 μm, “apoptotic bodies”, “oncosomes”, or “tumor microparticles”), intermedi-ate (0.2–1 μm, “microparticles”), to small (0.05–0.2 μm, “exo-somes”) tdEVs, EV-miRNAs, and ccfDNA.

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Model Validation

Figure 2 shows the modeled and measured bead recovery in PBS (top) and plasma (bottom). As expected, the recovery of beads in the pellet increases with increased particle diame-ter, g-force, and centrifugation time. In addition, because plasma is more viscous than PBS, the recovery of beads in the pellet is lower in plasma compared to PBS. Modeled and measured recovery are in fair agreement (all errors <8%) for both PBS and plasma at 300g and 2,700g, as well as for PBS at 15,800g. The model overestimates the particle speed at 15,800g for plasma, with a maximum error of 20%. Thus, the model is a reasonable approximation to predict the behavior of spherical particles when applied to the centrifugation pro-tocols as shown in Table 2.

Predicted Performance of Centrifugation Protocols Table 3 shows the predicted recovery of number of parti-cles based on the centrifugation protocols as shown in Table 2. The volume of the supernatant plus the volume of the pellet equals the starting volume. The volume reduction can be calculated by dividing the starting volume by the vol-ume of supernatant or pellet. To obtain the particle concen-tration after centrifugation, the predicted recovery in the number of particles needs to be multiplied with the volume reduction, which is shown in the last column of Table 3.

The CTC protocol recovers 100% of CTCs but co-isolates substantial fractions of TEPs, large tdEVs, and ccfDNA. In subsequent epithelial cell adhesion molecule (EpCAM)-based magnetic enrichment of CTCs, the TEPs and ccfDNA will be removed, but tdEVs exposing EpCAM will be co-isolated. The latter is confirmed by a previous study, in which the CTC fraction was shown to contain large EpCAM+ EVs (5).

The protocol to isolate TEPs predicts a yield of 71% together with 22% large tdEVs and <3% CTCs, smaller tdEVs, and ccfDNA. Because the subsequent processing includes detection of all mRNA present in the sample, our model indi-cates that additional evidence is needed to prove that the obtained RNA profiles indeed originate from platelets.

EV-miRNA samples contain intermediate tdEVs (40%), small tdEVs (57%), ccfDNA (57%), and TEPs and large tdEVs (<1%). By subsequent size exclusion chromatography (23), ccfDNA is removed, resulting in a relatively pure tdEV sample with high yield. The model predicts that the concen-tration of tdEVs is unaffected by the final centrifugation step

Figure 2. Centrifugation of beads diluted in PBS or plasma. The modeled bead recovery in pellet (blue dashed line) and supernatant (black, solid line) compared to measured bead recovery in pellet (open blue diamonds) and supernatant (closed black circles) for three centrifugation conditions. [Color figure can be viewed at wileyonlinelibrary.com]

of the protocol, which is confirmed by the authors upon inquiry (6).

For ccfDNA, two centrifugation protocols were evalu-ated. The Speicher protocol (8) predicts the 82% recovery of ccfDNA, together with large tdEVs (2%), intermediate tdEVs (63%), small tdEVs (82%), and negligible CTCs and TEPs. The Dawson protocol (7) yields 48% of ccfDNA, but with lower contamination of intermediate tdEVs (6%), small tdEVs (49%), and negligible CTCs, TEPs, and large tdEVs. Thus, both protocols do not yield pure ccfDNA. Because the volu-metric mass density difference between DNA and plasma is at least ninefold higher than the density difference for EVs, it is difficult to separate DNA from small EVs. Consequently, a 35 nm DNA “particle” travels at the same speed as a 100 nm EV.

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Centrifugation can be described with a model based on the Stokes equation. The model illustrates that centrifugation is effective at recovering particles that can be pelleted but that additional methods are needed to obtain a pure fraction. Par-ticles with similar sedimentation rates are always co-isolated during centrifugation. This is especially relevant for TEP and ccfDNA protocols, because the co-isolated tdEVs may have substantial impact on the outcome. This model offers a tool to (re)design centrifugation protocols and at a minimum to establish whether available candidate protocols differ suffi-ciently from each other to warrant comparison. The model includes a shape factor to account for the nonspherical shape of platelets. This shape factor was set to 2.0 based on litera-ture but was not experimentally validated.

For technologies evaluating the presence of CTCs, such as the CellSearch system, whole blood is centrifuged at 800g for 10 min. For this application, the model predicts a 100% effective isolation of CTCs, in addition to a recovery of 81% of the large tdEVs and 39% of TEPs. However, because of the use of imaging to identify each particle, tdEVs and TEPs can be distinguished from CTCs, and thus, the co-isolation does not affect the determined CTC concentration. Nevertheless, a purer CTC sample may be obtained by adjusting the

centrifugation protocol. To reduce the recovery of TEPs or intermediate size tdEVs, the g-force or centrifugation time should be reduced. Because of their size, ccfDNA or small tdEVs’ recovery in the pellet is mainly reduced by a reduction of the collection height.

The model predicts that a single biomarker cannot be purified by centrifugation alone. Substantial fractions of co-isolated other biomarkers remain present in all evaluated pro-tocols, which may affect the obtained results and interpreta-tion thereof.

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This work was supported by the Netherlands Organisa-tion for Scientific Research—Domain Applied and Engineer-ing Sciences (NWO-TTW), research programs VENI 13681 (FC), Perspectief CANCER-ID 14198 (LR), and VENI 15924 (EvdP).

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The authors declare that the research was carried out in the absence of any personal, professional, or financial rela-tionship that could potentially be construed as a conflict of interest.

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Table 3. Predicted recovery (%) of centrifugation protocols

PROTOCOL

CHANGE IN NUMBER (% OF ORIGINAL)

VOL. RED. CTCS TEPS LARGE TDEVS INTERMEDIATE TDEVS SMALL

TDEVS CCFDNA (FOLD)

CTCs/tdEVs 100.0 39.4 81.1 16.7 13.3 13.3 2.9 TEPs 0.0 71.1 21.7 3.2 2.2 2.2 3.0 EV-miRNA 0.0 0.01 0.7 39.7 57.0 56.9 1.7 ccfDNA (Speicher) 0.0 0.05 2.4 63.4 82.0 82.0 1.2 ccfDNA (Dawson) 0.0 6.10−7 0.0 6.0 49.0 48.4 1.7

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